Selectable (or selective) markers and agents are important for genetically modifying cells or microorganisms. They can be used to screen for cells with introduced DNA: A selectable marker protects the organism from the presence or absence of a selective agent that would normally kill it or prevent its growth. Usually antibiotic resistance genes are used as selectable markers, and the respective antibiotic as selective agent.
Use of selectable agents like antibiotics adds significant costs to a process like an industrial fermentation. Some antibiotics are also not or only poorly soluble in water and need to be dissolved in solvents. This adds further costs and potentially has a negative effect on the microbial cells; for example, chloramphenicol and thiamphenicol need to be dissolved in Ethanol or Dimethylformamide (DMF), respectively.
Some organisms are also naturally resistant to some antibiotics and so they can not be used as selectable agents. For example, most antibiotics classes are only active against either Gram-positive or Gram-negative bacteria as they target cell wall compounds and several Genera have natural resistance against specific antibiotics (for example, Clostridia against Chloramphenicol, as they posses chloramphenicol acetyltransferase genes that confer resistance and are also able to reduce the aryl-nitro-residue of the molecule to inactivate it (O'Brien & Morris, 1971, J Gen Microbiol, 67: 265-271)).
In addition, some antibiotic substances get inactivated or cannot be used under typical process conditions. For example, the low pH between 4-5.5 used in several fermentation processes inactivates the macrolide erythromycin, while on the other hand it's analogue clarithromycin only dissolves at extremely low pH below 2 (Mermelstein & Papoutsakis, 1993, FEMS Microbial Lea, 113: 71-76).
Auxotrophic markers that can compensate for an inability to metabolise certain amino acids, nucleotides, or sugars can also be used for selection. However, these also require the addition of compounds to the media which are not otherwise needed, increasing expense.
Reporter genes have also been used to allow for selection of successful transformants during processes for producing recombinant microorganisms; for example, genes encoding green fluorescent protein or beta-galactosidase (lacZ). However, these can be toxic to the cells and the products produced undesirable in commercial fermentation reactions.
It is an object of the invention to overcome one or more of the disadvantages of the prior art, or to at least to provide the public with a useful choice.